The proposed research involves detailed investigations of the relationship between the physical and chemical properties and the hormonal function of insulin. The primary objective of this project involves the investigation of the mechanism of assembly of the zinc-insulin hexamer, (In)6(Zn++)2, the storage form of insulin. Experiments using laser light scattering and uv-visible absorbance in combination with rapid-mixing stepped-flow spectrophotometry will be carried out to study (1) the assembly of the insulin subunits (as measured by changes in light scattering), (2) the uptake of Zn(II) and/or Ca(II) during assembly (using chromophoric chelators as indicators of the concentration of free metal ion), (3) the thermodynamics of metal ion binding and subunit assembly via equilibrium binding studies, and (4) the study of the physical and chemical properties of metal-ion-substituted insulin hexamers. The metal ions to be studied include Ca(II), Cd(II), Co(II), Cu(II), Co(III), Cr(III) and Tb(III). Our recent discovery that (In)6(Zn++)2 is a calcium binding protein (Sudmeier, J. L., Bell, S. J. Storm, M.C. and Dunn, M.F. (1981) Science 212, 560) make the study of Ca(II) effects on hexamer formation of special interest to this proposal. The Co(III) and Cr(III) derivatives may be of special interest as slow release forms of insulin (due to the exchange inert properties of these metal ions) for clinical use in the treatment of diabetes mellitus. It is anticipated that this study will provide insight into the understanding of the control mechanisms which regulate the storage and release of hexameric insulin and the formation of monomeric insulin, the active form.